The contribution of DNA and chromosome repair deficiencies to the radiosensitivity of ataxia-telangiectasia.

Cells derived from individuals with ataxia-telangiectasia (AT) are more sensitive to ionizing radiation and radiomimetic drugs, as evidenced by decreased survival and increased chromosome aberrations at mitosis when compared with normal cell lines. Our previous studies showed that, despite similar initial levels of DNA double-strand breaks (DSBs), AT cells express higher initial chromosome damage than do normal cells as demonstrated by the technique of premature chromosome condensation. However, this finding accounted for only a portion of the increased sensitivity (T. K. Pandita and W. N. Hittelman, Radiat. Res. 130, 94-103, 1992). The purpose of the study reported here was to examine the contribution of DNA and chromosome repair to the radiosensitivity of AT cells. Exponentially growing AT and normal lymphoblastoid cells were fractionated into cell cycle phase-enriched populations by centrifugal elutriation, and their DNA and chromosome repair characteristics were evaluated by DNA neutral filter elution (for DNA DSBs) and by premature chromosome condensation, respectively. AT cells exhibited a reduced fast-repair component in both G1- and G2-phase cells, as observed at the level of both DNA DSBs and the chromosome; however, S-phase cells showed nearly normal DNA DSB repair. The findings that AT cells exhibit an increased level of chromosome damage and a deficiency in the fast component (but not the slow component) of repair suggest that chromatin organization might play a major role in the observed sensitivity of AT cells. When survival was plotted as a function of the residual amount of chromosome damage in G1- and G2- phase cells after 90 min of repair, the curves for normal and AT cells approached each other but did not overlap. These results suggest that, although higher initial levels of chromosome damage and reduced chromosome repair capability can explain much of the radiosensitivity of AT cells, other differences in AT cells must also contribute to their sensitivity phenotype.     

The relationship of DNA and chromosome damage to survival of synchronized X-irradiated L5178Y cells. I. Initial damage

We investigated the role of initial DNA and chromosome damage in determining the radiosensitivity difference between the variant murine leukemic lymphoblast cell lines L5178Y-S (sensitive) and L5178Y-R (resistant) and the difference in cell cycle-dependent variations in radiosensitivity of L5178Y-S cells. We measured initial DNA damage (by the neutral filter elution method) and chromosome damage (by the premature chromosome condensation method) and compared them with survival (measured by cloning) for both cell lines synchronized in G1 or G2 phase of the cell cycle (by centrifugal elutriation) and irradiated with low doses of X rays (up to 10 Gy). The initial yield of DNA and chromosome damage in G2 L5178Y-S cells was almost twice that in G1 L5178Y-S cells and G1 or G2 L5178Y-R cells. In all cases DNA damage expressed as relative elution corresponded with chromosome damage (breaks in G1 chromosomes, breaks and gaps in G2 chromosomes). Also we found that the initial DNA and chromosome damage did not determine cell age-dependent radiosensitivity variations in L5178Y-S cells, as there was less initial damage in the more sensitive G1 phase than in the G2 phase. L5178Y-R cells showed only small changes in survival or initial yield of DNA and chromosome damage throughout the cell cycle. Because survival and initial damage in sensitive and resistant cells irradiated in G2 phase correlated, the difference in radiosensitivity between L5178Y-S and L5178Y-R cells might be determined by initial damage in G2 phase only.     

Mechanism of radiosensitization by halogenated pyrimidines: effect of BrdU on radiation induction of DNA and chromosome damage and its correlation with cell killing

The effect of BrdU incorporation on cell radiosensitivity as well as on the induction of DNA double-strand breaks (DSB) and chromosome damage by radiation was studied in CHO cells. Induction of DNA DSB was measured by the nonunwinding filter elution technique and damage at the chromosome level was visualized and scored in G1 cells using the technique of premature chromosome condensation. The results indicated an increase in the radiosensitivity of cells grown in the presence of BrdU. Although sensitization was observed both in cells irradiated in the exponential phase and in cells irradiated in the plateau phase of growth, the degree of sensitization was greater in exponentially growing cells for the same degree of thymidine replacement by BrdU in the DNA. It is hypothesized that this indicates the possible importance of chromatin structure at the time of irradiation and/or the importance of chromatin conformation changes after irradiation in the expression of radiation-induced potentially lethal damage in cells containing BrdU. Incorporation of BrdU affected both the slope and the width of the shoulder of the survival curve and increased the induction of DNA and chromosome damage per unit absorbed dose. The increase observed in the slope of the survival curve was quantitatively similar to the increase observed in damage induction at the DNA and the chromosome level, suggesting a cause-effect relationship between these phenomena. Reduction in the width of the shoulder did not correlate with the increase in the induction of DNA and chromosome damage, suggesting that different phenomena, probably related to enhanced fixation of radiation-induced potentially lethal damage in cells containing BrdU, underlie its modulation.     

Induction of chromosome damage by Neurospora endonuclease in repair-inhibited quiescent normal human fibroblasts

The purpose of these experiments was to determine the role of double-strand breaks in chromosome aberration formations. Quiescent normal human fibroblasts were treated with 3 μM nitrogen mustard and then allowed to repair their DNA damage for 24 h prior to cell fusion and induction of premature chromosome condensation. The extent of chromosome damage was determined in the G1 prematurely condensed chromosomes (G1 PCC). The presence of cytosine arabinoside and hydroxyurea during the repair period in order to accumulate single-strand DNA breaks resulted in an increase in the chromosome-break frequency. Treatment of these repair-inhibited cells with single-strand-specific neurospora endonuclease during fusion to change single-strand lesions into double-strand breajs resulted in a doubling of the aberration frequency. These results support the notion that double-strand breaks are important in chromosome-aberration formation.     

The relationship of DNA and chromosome damage to survival of synchronized X-irradiated L5178Y cells. II. Repair

The purpose of this study was to investigate the role of DNA and chromosome repair in determining the difference in radiosensitivity between a radiosensitive murine leukemic lymphoblastoid cell line, L5178Y-S, and its radioresistant counterpart, L5178Y-R. Populations of cells in the G1 or G2 phase of the cell cycle were obtained by centrifugal elutriation and irradiated with X-ray doses up to 10 Gy and allowed to repair at 37 degrees C for various periods. The kinetics of DNA double-strand break repair was estimated using the DNA neutral filter elution method, and the kinetics of chromosome repair was measured by premature chromosome condensation. L5178Y-S cells exhibited decreased repair rates and limited repair capacity at both the DNA and chromosome level in both G1 and G2 phases when compared to L5178Y-R cells. For the repair-competent L5178Y-R cells, the rate of DNA repair was similar in G1 and G2 cells and exhibited both fast and slow components. While the kinetics of chromosome break repair in G1 cells was similar to that of DNA repair, chromosome repair in G2 cells had a diminished fast component and lagged behind DNA repair in terms of fraction of damage repaired. Interestingly, concomitant with a diminished repair capacity in L5178Y-S cells, the number of chromatid exchanges in G2 cells increased with time, whereas it remained constant with repair time in L5178Y-R cells. These results suggest that the basis for the exceptional radiosensitivity of L5178Y-S cells is a defect in the repair of both DNA double-strand breaks and chromosome damage.     

Gamma-irradiation and doxorubicin treatment of normal human cells cause cell cycle arrest via different pathways

Ionizing radiation and doxorubicin both produce oxidative damage and double-strand breaks in DNA. Double-strand breaks and oxidative damage are highly toxic and cause cell cycle arrest, provoking DNA repair and apoptosis in cancer cell lines. To investigate the response of normal human cells to agents causing oxidative damage, we monitored alterations in gene expression in F65 normal human fibroblasts. Treatment with g-irradiation and doxorubicin altered the expression of 23 and 68 known genes, respectively, with no genes in common. Both agents altered the expression of genes involved in cell cycle arrest, and arrested the treated cells in G2/M phase 12 h after treatment. 24 h after g-irradiation, the percentage of G1 cells increased, whereas after doxorubicin treatment the percentage of G2/M cells remained constant for 24 h. Our results suggest that F65 cells respond differently to g-irradiation- and doxorubicin-induced DNA damage, probably using entirely different biochemical pathways.     

Hypersensitivity of lymphoblastoid lines derived from ataxia telangiectasia patients to the induction of chromosomal aberrations by etoposide (VP-16)

Mammalian DNA topoisomerase II represents the cellular target of many antitumor drugs, such as epipodophyllotoxin VP-16 (etoposide). The mechanism by which VP-16 exerts its cytotoxic and antineoplastic actions has not yet been firmly established, although the unique correlation between sensitivity to ionizing radiation and to topoisomerase II inhibitors suggest the involvement of DNA double-strand breaks. In the present study we analyzed the chromosomal sensitivity of lymphoblastoid cell lines derived from ataxia telangiectasia (AT) patients to low concentrations of the drug. Our results indicate that AT derived cells are hypersensitive to the clastogenic activity of VP-16 either when the drug is present for the whole duration of the cell cycle or specifically in the G2 phase, confirming that the induction of DNA double strand breaks, to which AT cells seem typically sensitive, could have an important role in the biological activity of VP-16.     

DNA double-strand damage and repair following gamma-irradiation in isolated spermatogenic cells

Various cell types in spermatogenesis exhibit differential sensitivity to radiation-induced DNA damage. The investigation of DNA radiosensitivity in vitro is complicated by the heterogeneous population of male germ cells (MGC) present in isolated single-cell suspensions. In the present investigation, the neutral elution technique was used to assess gamma-irradiation-induced DNA double-strand damage (DSD) in spermatogonia and preleptotene spermatocytes (SG/PL), pachytene spermatocytes and spermatid spermatocytes, as well as in MGC. In addition, the capability of these cell types to repair DNA double-strand damage was investigated. Based on the well established timing of the rat spermatogenic cycle, the DNA of specific cell populations was labeled using tritiated thymidine. DNA from labeled cells was determined isotopically, whereas total DNA was quantitated using a fluorometric method. DSD was induced in a dose-dependent manner in the heterogeneous population as well as in the labeled cell populations. SG/PL were more sensitive to gamma-irradiation-induced DSD than either the heterogeneous MGC population, pachytene or spermatid spermatocytes. Each cell type exhibited a similar capability to repair DSD following exposure to 3000 rad; repair was rapid (maximal within 45 min) and incomplete (less than 40%). Only pachytene spermatocytes exhibited significant repair following exposure to 6000 rad. Since a difference in sensitivity to radiation-induced DSD was demonstrated, the capability of each cell type to repair a similar initial frequency of strand damage was investigated. SG/PL, pachytene and spermatid spermatocytes differed in their capability to repair similar levels of strand damage. However, the difference in dose required to achieve equal damage may have contributed to other cellular effects, thus altering repair. In summary, a model is described that permits the evaluation of genotoxic responses in specific populations of spermatogenic cells within a heterogeneous cell suspension. The ability of specific cell types to repair gamma-irradiation-induced DNA double-strand damage is demonstrated.     

Interaction between radiation and drug damage in mammalian cells. V. DNA damage and repair induced in LZ cells by adriamycin and/or radiation

A multi-drug-resistant cell line selected in increasing concentrations of Adriamycin and designated LZ (J. A. Belli, Radiat. Res. 119, 88-100, 1989) is shown to exhibit a survival response characterized by radiation sensitivity and Adriamycin resistance. To determine if this response is due to alterations in either the initial levels of damage induced or the repair of DNA damage, LZ cells and the parental V79 cells were exposed to either radiation or Adriamycin and the damage and repair were measured with alkaline or nondenaturing filter elution. After exposure to radiation, induction and repair of both single-strand and double-strand breaks were equivalent. LZ cells exposed to 100 micrograms/ml Adriamycin for 1 h contained no measurable damage while the same treatment induced breaks and crosslinks in V79 cells. Pretreatment of LZ cells for 1 h with Adriamycin before irradiation did not alter either the initial levels of induced damage or the repair of strand breakage. These results suggest that (1) mechanisms other than differential induction and repair of strand breaks are responsible for the increased radiation sensitivity in LZ, and (2) the lack of Adriamycin-induced DNA damage in LZ is at least partially responsible for the increased cell survival after treatment.     

Influence of double-strand-break repair pathways on radiosensitivity throughout the cell cycle in CHO cells

Unrepaired DNA double-strand breaks (DSBs) produced by ionizing radiation (IR) are a major determinant of cell killing. To determine the contribution of DNA repair pathways to the well-established cell cycle variation in IR sensitivity, we compared the radiosensitivity of wild-type CHO cells to mutant lines defective in nonhomologous end joining (NHEJ), homologous recombination repair (HRR), and the Fanconi anemia pathway. Cells were irradiated with IR doses that killed approximately 90% of each asynchronous population, separated into synchronous fractions by centrifugal elutriation, and assayed for survival (colony formation). Wild-type cells had lowest resistance in early G1 and highest resistance in S phase, followed by declining resistance as cells move into G2/M. In contrast, HR-defective cells (xrcc3 mutation) were most resistant in early G1 and became progressively less resistant in S and G2/M, indicating that the S-phase resistance in wild-type cells requires HRR. Cells defective in NHEJ (dna-pk(cs) mutation) were exquisitely sensitive in early G1, most resistant in S phase, and then somewhat less resistant in G2/M. Fancg mutant cells had almost normal IR sensitivity and normal cell cycle dependence, suggesting that Fancg contributes modestly to survival and in a manner that is independent of cell cycle position.     

Chromatid damage induced by fluorescent light during G2 phase in normal and Gardner syndrome fibroblasts. Interpretation in terms of deficient DNA repair

Skin fibroblasts from Gardner syndrome (GS) compared with those from normal donors showed a significantly higher incidence of chromatid gaps and breaks following exposure to low-intensity, cool-white fluorescent light during G2 phase of the cell cycle. Considerable evidence supports the concept that chromatid gaps and breaks seen directly after exposure to DNA-damaging agents represent unrepaired DNA single- and double-strand breaks respectively. The changes in incidence of chromatid aberrations with time after light exposure are consistent with the sequence of events known to follow DNA damage and repair. Initially, the incidence of light-induced chromatid gaps was equivalent in GS and normal fibroblasts. In the normal cells, the chromatid gaps disappeared by 1 h post-exposure, presumably as a result of efficient repair of DNA single-strand breaks. In contrast, the incidence of gaps increased in GS cells by 0.5 h followed by a decrease at 1 h and concomitant increase in chromatid breaks. It appears from these findings that the increased incidence of chromatid damage in GS fibroblasts results from deficient repair of DNA single-strand breaks which arise from incomplete nucleotide excision of DNA damage during G2 phase.     

Histone deacetylase 4 interacts with 53BP1 to mediate the DNA damage response

Anumber of proteins are recruited to nuclear foci upon exposure to double-strand DNA damage, including 53BP1 and Rad51, but the precise role of these DNA damage-induced foci remain unclear. Here we show in a variety of human cell lines that histone deacetylase (HDAC) 4 is recruited to foci with kinetics similar to, and colocalizes with, 53BP1 after exposure to agents causing double-stranded DNA breaks. HDAC4 foci gradually disappeared in repair-proficient cells but persisted in repair-deficient cell lines or cells irradiated with a lethal dose, suggesting that resolution of HDAC4 foci is linked to repair. Silencing of HDAC4 via RNA interference surprisingly also decreased levels of 53BP1 protein, abrogated the DNA damage-induced G2 delay, and radiosensitized HeLa cells. Our combined results suggest that HDAC4 is a critical component of the DNA damage response pathway that acts through 53BP1 and perhaps contributes in maintaining the G2 cell cycle checkpoint.     

Cell sorting analysis of cell cycle-dependent X-ray sensitivity in end joining-deficient human cells

Non-homologous end joining (NHEJ) plays a major role in the repair of ionizing radiation-induced DNA double-strand breaks (DSBs), especially during the G1-phase of the cell cycle. Using a flow cytometric cell sorter, we fractionated G1- and S/G2-phase cells based on size to assess the DSB-repair activity in NHEJ factor-deficient DT40 and Nalm-6 cell lines. Colony formation assays revealed that the X-ray sensitivities of the G1-enriched populations correctly reflected the DSB-repair activities of both the DT40 and Nalm-6 cell lines. Furthermore, as assessed by γ-H2AX foci formation, the sorted cells exhibited less DNA damage than chemically synchronized cells. Given that it does not use fluorescent labeling or chemical agents, this method of cell sorting is simpler and less toxic than other methods, making it applicable to a variety of cell lines, including those that cannot be synchronized by standard chemical treatments.     

The repair of double-strand DNA breaks correlates with radiosensitivity of L5178Y-S and L5178Y-R cells

To better understand the basis for the difference in radiosensitivity between the variant murine leukemic lymphoblast cell lines L5178Y-R (resistant) and L5178Y-S (sensitive), the production and repair of DNA damage after X irradiation were measured by the DNA alkaline and neutral elution techniques. The initial yield of single-strand DNA breaks and the rates of their repair were found to be the same in both cell lines by the DNA alkaline elution technique. Using the technique of neutral DNA elution, L5178Y-S cells exhibited slightly increased double-strand breakage immediately after irradiation, most significantly at lower doses (i.e., less than 10 Gy). Nevertheless, even at doses that yielded equal initial double-strand breakage of both cell lines, the survival of L5178Y-S cells was significantly less than that of L5178Y-R cells. When the technique of neutral DNA elution was employed to measure the kinetics of DNA double-strand break repair, both cell lines exhibited biphasic fast and slow components of repair. However, the double-strand repair rate was much lower in the radiosensitive L5178Y-S cells than in the L5178Y-R cells (T1/2 of 60 vs 16 min). This difference was more pronounced in the fast-repair component. These results suggest that the repair of double-strand DNA breaks is an important factor determining the radiosensitivity of L5178Y cells.     

Cell cycle effect on the induction of DNA double-strand breaks by X rays

Filter elution was used to compare X-ray-induced DNA single- and double-strand breaks in proliferating (P) and quiescent (Q) cells of the 66 and 67 mouse mammary tumor lines. There was no difference either between cell type or between growth states in the amount of single-strand breaks as defined by elution at pH 12.2. In contrast, Q cells appeared to sustain a much larger amount of double-strand break damage per Gray than P cells, when the damage was measured by elution at either pH 7.2 or pH 9.6. Experiments which combined centrifugal elutriation with pH 7.2 elution demonstrated that G1-P cells were similar to Q (greater than or equal to 95% G1) cells in the induction of elution-detectable double-strand breaks, while the S-phase enriched fractions sustained less damage than G1-P, Q, or asynchronous P populations. Studies in which P populations were pulse labeled with [14C]thymidine confirmed this finding. Mathematical analysis of the elution kinetics of irradiated P, Q, and S-phase cells supports a model in which the complex elution profiles observed for P cells could be explained as the sum of the one-component exponential elution profiles of G1- and S-phase subpopulations. Also, the correlation between damage measured by pH 7.2 elution and cell survival was tested by examining the dose response for stimulated 66 cells (St4), which like Q cells are greater than or equal to 95% in G1 but are more resistant to X-ray-induced cytotoxicity than are the 66 Q cells. However, the induction of double-strand breaks in St4 cells was identical to that in Q cells. Thus we conclude that there is not necessarily a correlation between the amount of elution-detectable X-ray-induced double-strand breaks and cell survival.     

Induction by H2O2 of DNA and interphase chromosome damage in plateau-phase Chinese hamster ovary cells.

The induction by H2O2 of DNA breaks, DNA double-strand breaks (DSBs), and interphase chromatin damage and their relationship to cytotoxicity were studied in plateau-phase Chinese hamster ovary (CHO) cells. Damage in interphase chromatin was assayed by means of premature chromosome condensation (PCC); DNA DSBs were assayed by nondenaturing filter elution (pH 9.6), and DNA breaks by hydroxyapatite chromatography. Cells were treated with H2O2 in suspension at 0 degrees C for 30 min and treatment was terminated by the addition of catalase. Concentrations of H2O2 lower than 1 mM were not cytotoxic, whereas concentrations of 40 and 60 mM reduced cell survival to 0.1 and 0.004, respectively. An induction of DNA breaks that was dependent on H2O2 concentration was observed at low H2O2 concentrations that reached a maximum at approximately 1 mM; at higher H2O2 concentrations induction of DNA breaks either remained unchanged or decreased. Damage at the chromosome level was not evenly distributed among the cells, when compared to that expected based on a Poisson distribution. Three categories of cells were identified after exposure to H2O2: cells with intact, control-like chromosomes, cells showing chromosome fragmentation similar to that observed in cells exposed to ionizing radiation, and cells showing a loss in the ability of their chromatin to condense into chromosomes under the PCC reaction. The fraction of cells with fragmented chromosomes, as well as the number of excess chromosomes per cell, showed a dose response similar to that of DNA DSBs, reaching a maximum at 1 mM and decreasing at higher concentrations. The results indicate that induction of DNA and chromosome damage by H2O2 follows a complex dependence probably resulting from a depletion of reducing equivalents in the vicinity of the DNA. Reducing equivalents are required to recycle the transition metal ions that are needed to maintain a Fenton-type reaction. The absence of cell killing at H2O2 concentrations that yielded the maximum amount of DNA and chromosome damage suggests that this damage is nonlethal and repairable. It is suggested that lethal DNA and chromosome damage is induced at higher concentrations of H2O2 where cell killing is observed by an unidentified mechanism.     

Mechanism of radiosensitization by halogenated pyrimidines: effect of BrdU on repair of DNA breaks, interphase chromatin breaks, and potentially lethal damage in plateau-phase CHO cells.

There is evidence suggesting that radiosensitization induced in mammalian cells by substitution in the DNA of thymidine with BrdU has a component that relies on inhibition of repair and/or fixation of radiation damage. Here, experiments designed to study the mechanism of this phenomenon are described. The effect of BrdU incorporation into DNA was studied on cellular repair capability, rejoining of interphase chromosome breaks, as well as induction and rejoining of DNA double- and single-stranded breaks (DSBs and SSBs) in plateau-phase CHO cells exposed to X rays. Repair of potentially lethal damage (PLD), as measured by delayed plating of plateau-phase cells, was used to assay cellular repair capacity. Rejoining of interphase chromosome breaks was assayed by means of premature chromosome condensation (PCC); induction and rejoining of DNA DSBs were assayed by pulsed-field gel electrophoresis and induction and rejoining of DNA SSBs by DNA unwinding. A decrease was observed in the rate of repair of PLD in cells grown in the presence of BrdU, the magnitude of which depended upon the degree of thymidine replacement. The relative increase in survival caused by PLD repair was larger in cells substituted with BrdU and led to a partial loss of the radiosensitizing effect compared to cells tested immediately after irradiation. A decrease was also observed in the rate of rejoining of interphase chromosome breaks as well as in the rate of rejoining of the slow component of DNA DSBs in cells substituted with BrdU. The time constants measured for the rejoining of the slow component of DNA DSBs and of interphase chromosome breaks were similar both in the presence and in the absence of BrdU, suggesting a correlation between this subset of DNA lesions and interphase chromosome breaks. It is proposed that a larger proportion of radiation-induced potentially lethal lesions becomes lethal in cells grown in the presence of BrdU. Potentially lethal lesions are fixed via interaction with processes associated with cell cycle progression in cells plated immediately after irradiation, but can be partly repaired in cells kept in the plateau-phase. It is hypothesized that fixation of PLD is caused by alterations in chromatin conformation that occur during normal progression of cells throughout the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction between radiation and drug damage in mammalian cells. VI. Radiation and doxorubicin age-response function of doxorubicin-sensitive and -resistant Chinese hamster cells.

A comparative study of the radiation and/or doxorubicin (DOX) survival response for synchronous populations of Chinese hamster V79 cells and two DOX-resistant variants (77A and LZ-8) was performed. The greatest cellular radiation sensitivity was observed in mitosis, while the greatest resistance was observed during late S phase for the three cell lines. The variation in radiation response throughout the cell cycle was expressed as a change in the width of the shoulder of the survival curves (Dq) with little change in D0. This suggests that each phase of the cell cycle has a different capacity for accumulation of radiation injury. The radiation age-response function for the three cell lines revealed that 77A and LZ-8 cells were more radiosensitive than V79 cells throughout the cell cycle. Exposure of synchronous populations to DOX (1.84 microM for V79, 9.21 microM for 77A, and 921 microM for LZ-8) for 1 h as a function of cell cycle phase revealed that V79, 77A, and LZ-8 cells exhibited the greatest sensitivity to DOX in mitosis and the most resistance to DOX during S phase, as indicated by the differences in the slope of the initial component of the survival curve. Levels of P-glyco-protein (P-gp) are probably not a factor contributing to DOX age-response function since P-gp levels remain constant throughout the cell cycle in all three cell lines. Synchronous populations of V79, 77A, and LZ-8 cells sequentially treated with DOX and radiation at various cell cycle phases were also analyzed. The results showed that the interaction between radiation and DOX damage resulted in a reduced cellular capacity for the accumulation of radiation damage throughout the cell cycle, as indicated by a decrease in the width of the shoulder of the survival curve. Overall, both DOX-sensitive V79 cells and DOX-resistant 77A and LZ-8 cells exhibited (1) a similar age-response function for radiation or DOX, and (2) no differences in the effects of DOX on radiation-induced damage throughout the cell cycle. These results indicate that acquired resistance to DOX associated with increased levels of P-gp in the cell membrane did not appear to affect the age-response function for radiation or DOX, and the nature of the interaction between damage caused by radiation and DOX was also not affected.     

The effects of hyperthermia and ionizing radiation in normal and ataxia telangiectasia human fibroblast lines

The effects of 45 degrees C hyperthermia and gamma radiation have been studied in three normal human fibroblast lines (GM38, GM730, WI38) and compared to the effects in two lines derived from patients with the hereditary disease ataxia telangiectasia (AT3BI, AT5BI). All lines, both normal and gamma-sensitive AT, showed a similar resistance to killing by heat alone, suggesting that the defect responsible for the increased radiation sensitivity in AT lines does not confer increased heat sensitivity. Shouldered survival curves were obtained in each case indicating the ability to accumulate sublethal heat damage. All normal and AT cell lines exhibited increased resistance to the lethal effects of heat in response to a thermal stress, indicating that the defect that causes radiosensitivity in AT cell lines does not prevent the induction of thermotolerance. Heat (45 degrees C, 30 min) was shown to increase the sensitivity of the normal cell lines to killing by gamma radiation. The thermal enhancement ratios obtained ranged from about 2.5 to 3.0. The same heat treatment, however, produced very little increase in the radiation sensitivity of the AT cells. Thermal enhancement ratios of about 1.2 were obtained in these lines. We hypothesize that, in normal cells, this heat treatment inactivates the process which is already defective in AT lines, and that this process may be required for the proper rejoining of double-strand breaks produced during the repair of other radiation-induced lesions.     

The radiosensitivity of the chromosomes of the cells of human squamous cell carcinoma cell lines.

Measurement of the radiation sensitivity of chromosomes was used to address the influence of cell cycle distribution and of DNA content and ploidy on radiation responses in seven human squamous cell carcinoma cell lines. The cell lines varied about twofold in DNA content and chromosome number, and the X-ray sensitivities (D0) of the lines ranged from 1.1 to 2.7 Gy. The more resistant cell lines (D0 greater than 1.8 Gy) had faster growth rates and larger proportions of cells in S phase in asynchronous cultures. Aberration frequencies were measured in cells irradiated in G1 and G2 phase. The more resistant lines had fewer induced aberrations in both phases than did sensitive lines, implying that they were more resistant to radiation in both of these cell cycle phases. Therefore, while the larger S-phase population seen in the resistant cell lines probably contributes to the resistant phenotype, it cannot explain all of the intrinsic differences in radiation sensitivity. There was no relationship between DNA content and radiation sensitivity as measured by the cell survival assay or the induction of chromosome aberrations, although cells with larger DNA contents tended to have more chromosome damage per cell at equitoxic doses.